The present invention relates generally to the field of detection assays and, more particularly, to fluorescent quenching methods and compositions for quantifying the extent of ligand pair binding on solid supports, for example those employed in nucleic acid hybridizations.
Nucleic acid hybridization is a known method for identifying specific sequences of nucleic acids. Hybridization is based upon base pairing between complementary nucleic acid strands. When single stranded nucleic acids are incubated in appropriate buffer solutions, complementary base sequences pair to form double stranded stable molecules. The presence or absence of such pairing may be detected by several different methods described in the art.
Hybridization assays generally involve multiple steps, for example the hybridization technique described by Dunn, et al., Cell 12:23-36 (1977) (incorporated herein by reference), wherein a sandwich-type assay consists of a first hybridization between a "target" nucleic acid and a "capture" nucleic acid probe that has been immobilized on a solid support and a second hybridization between a "signal" nucleic acid probe, typically labeled with a radioactive isotope, and a different region of the immobilized target nucleic acid. The hybridization of the signal probe may then be detected by, for example, autoradiography.
Ranki, et al., U.S. Pat. No. 4,486,539 and U.S. Pat. No. 4,563,419 (both patents incorporated herein by reference), describe sandwich-type assays that first require steps to render nucleic acids single stranded before the single stranded nucleic acids are allowed to hybridize with a nucleic acid affixed to a solid carrier and with a nucleic acid labeled with a radioisotope.
Litman, et al., U.S. Pat. No. 4,391,904 (incorporated herein by reference), describes test strip kits wherein a member of an immunological pair is bonded to a solid surface. Also, Miller, et al., Clin. Chem. 30:1467-1472 (1984), and Brown, et al., Clin. Chem. 31:1500-1505 (1985) (both articles incorporated herein by reference), describe an analytical test chamber containing cellulose threads coupled to an antibody as a solid matrix that permits multiple test results from a single sample.
An indirect quenching fluoroimmunoassay, double receptor fluoroimmunoassay, and protection fluoroimmunoassay represent assay formats in which antibodies are typically directed against the fluorescer. The assay is based on competition for the labeled antigen by the analyte-specific antibody and by the antibody directed against the fluorescer. See Hemmila, Clin. Chem. 31:359-370 (1985) (incorporated herein by reference) for a review of fluoroimmunoassays and immunofluorometric assays. See also Kobayashi, Steroids 36:177-183 (1980) (fluorescence quenching immunoassay of serum cortisol) (incorporated herein by reference).
Zuk, et al., U.S. Pat. No. 4,654,300 describes an immunoassay having conjugated fluorescent particles and conjugated catalyst wherein the particles and catalysts are conjugated to members of a specific binding pair.